The epidermis consists of a distinct cell layers that work in concert to execute a wide variety of essential functions. Keratinocytes comprise the majority of the cells of the epidermis, and form distinct strata from the basal proliferative layerto the outermost cornified layer. Keratinocytes of all strata secrete a variety of protein factors tha have critical roles in the proper function of the epidermis, including structural proteins, antimicrobial peptides, proteases and protease inhibitors, and cell-cell signaling molecules. Despite the recognized role of extracellular proteins in epidermal homeostasis, a comprehensive evaluation of these secreted factors-the keratinocyte secretome- has not been defined. The proposal describes the first comprehensive catalog of secreted epidermal proteins as identified by mass spectrometry. Analysis of the secretome of progenitor and differentiated keratinocytes reveals a number of novel factors whose function in the epidermis is unknown. Preliminary experiments using genetic depletion of candidate factors revealed a novel essential roles for the proteins calmodulin-like 5 (CALML5) and suprabasin (SBSN) in epidermal differentiation. The first aim of this proposal describes the use of genomic and genetic techniques, including analysis of whole transcriptome data from CALML5-depleted tissue and gene set enrichment analysis, to predict and analyze the role of CALML5 among known epidermal regulators. Next, the functional regions of CALML5 are determined using mutagenesis in a genetic rescue assay. [The second aim focuses on the function of SBSN, a protein expressed in differentiated epidermal tissue that is produced in three distinct isoforms. These isoforms are differentially enriched in the intracellular and extracellular compartments, raising the possibility that they may have discrete functions inside and outside the cell. Genetic experiments are proposed to define which isoform(s) are required for differentiation, and to determine the subcellular compartments that these isoforms are biologically active.] Together, the aims of this project are to discover and characterize new essential secreted regulators of skin homeostasis, which can provide a deeper understanding to the mechanisms of skin development and reveal new potential targets to treat skin disease.